LOCUS       sequence-486258-        6651 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Encodes human PIPK-gamma-87-D253A and N-terminal EBFP2 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6651)
  AUTHORS   Lee WN, Li J, Chen NP, Yu CH
  TITLE     Kindlin Regulates Mechanosensitive Activation and Adhesion Assembly 
            of Integrin beta6.
  JOURNAL   Adv Sci (Weinh). 2025 Jun 10:e01078. doi: 10.1002/advs.202501078.
  PUBMED    40492509
REFERENCE   2  (bases 1 to 6651)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Adv Sci 
            (Weinh). 2025 Jun 10:e01078. doi: 10.1002/advs.202501078."
COMMENT     Sequence Label: EBFP2-PIPK-gamma-87-D253A
FEATURES             Location/Qualifiers
     source          1..6651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        764..1067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1068..1271
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1222..1242
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1310..1319
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1316..1318,1319..1321,1322..2032)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1361..1382)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4142..4263
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4179..4198)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4233..4252
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4270..4725)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4407..4428)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4619..4638
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4752..4856
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4858..5215
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4858..5049
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4881..4901)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5066..5201
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5250..6044
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5250..6044
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5304..5323)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5914..5933
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6232..6251)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6276..6323
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
      721 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      781 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      841 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      901 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      961 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1021 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1081 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1141 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1201 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1261 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt
     1321 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1381 cgtaaacggc cacaagttca gcgtgagggg cgagggcgag ggcgatgcca ccaacggcaa
     1441 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1501 gaccaccctg agccacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
     1561 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1621 ggacgacggc acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctagtgaa
     1681 ccgcatcgag ctgaagggcg tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1741 ggagtacaac ttcaacagcc acaacatcta tatcatggcc gtcaagcaga agaacggcat
     1801 caaggtgaac ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc tcgccgacca
     1861 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct
     1921 gagcacccag tccgtgctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1981 ggagttccgc accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact
     2041 cagatctcga gctcaaagct tcgaattcat ggagctggag gtaccggacg aggcggagag
     2101 cgctgaggcg ggggccgtgc cctcggaggc ggcgtgggcg gcagagagcg gggcggcggc
     2161 aggtttggct cagaagaagg cggccccaac agaggttctg tccatgacgg cacagccggg
     2221 ccctggccat gggaagaagt tgggccatcg aggtgtggac gcatccggcg aaaccaccta
     2281 caagaagacc acctcctcca ccctgaaggg tgccatccag ctgggcatcg gctacaccgt
     2341 gggccacctg agctccaagc ccgaacgcga cgtgctcatg caggacttct acgtggtgga
     2401 gagcatcttc ttccccagcg aaggcagcaa cctcaccccc gcccaccact tccaggactt
     2461 ccgcttcaag acctatgcac ctgtcgcctt ccgctacttc cgggagctct ttgggatccg
     2521 gccagatgat tacttgtact ccctgtgcaa tgagccgctg atcgagctgt ccaacccggg
     2581 cgccagtggc tccctcttct acgtcaccag cgacgacgag ttcatcatca agaccgtcat
     2641 gcacaaggag gccgagttcc tgcagaagct gctccctggc tactacatga acctcaacca
     2701 gaacccgcgg acgctgctgc ccaagttcta tgggctgtac tgcgtgcagt cggggggcaa
     2761 gaacatccgc gtcgtggtca tgaacaacat cctgccccgc gtggtcaaga tgcacctcaa
     2821 gttcgccctc aagggctcca cctacaagcg gcgcgccagc aagaaggaga aggagaagag
     2881 cttccccacc tacaaggacc tggacttcat gcaggacatg cccgaggggc tcctgctgga
     2941 cgccgacacc ttcagcgccc tggtcaagac gctgcagcgg gactgcctgg tcctggaaag
     3001 tttcaagatc atggactaca gcctgctgct gggcgtgcac aacatcgacc agcacgagcg
     3061 cgagcggcag gcgcagggcg cccagagcac ctcagatgag aagcggcctg tgggccagaa
     3121 ggcgctctac tccacggcca tggagtccat ccagggtggc gccgcgcgcg gggaggccat
     3181 cgaatcggat gacacgatgg gcgggatccc cgctgtgaac ggccgcgggg agcggctgct
     3241 gctgcacatt ggcatcatcg acatcctgca gtcctacagg ttcatcaaga aactggagca
     3301 cacctggaag gccctcgtcc acgatgggga cacggtgtcc gtccaccgcc ccagcttcta
     3361 tgccgagcgc tttttcaagt tcatgagcaa cacggtcttt cggaagaact cctccctgaa
     3421 gtcctcgccc tccaagaagg ggcgcggcgg agccttgcta gctgtgaaac cgctggggcc
     3481 caccgctgcc ttctcggcca gccagatccc tagcgagcgg gaggaggccc agtacgacct
     3541 gcggggggcc cgcagctacc ccacgctgga ggacgaaggc cggcccgacc tcctgccctg
     3601 cacgccacct tctttcgaag aagccactac agcctccatt gccacgactc tgtcatccac
     3661 atccctctcc attcctgagc ggtccccctc ggagacgtcg gagcagccgc ggtacaggcg
     3721 gcgcacacag tcgtctggac aggatggcag gccgcaggag gagccacccg cggaagagga
     3781 tctgcagcag attacagtgc aggtggagcc tgcgtgcagc gtggagattg tggtccccaa
     3841 agaggaggac gcaggggtgg aggcttcccc ggccggtgcc tctgctgctg ttgaagtaga
     3901 aactgccagc caggcctcag acgaggaggg cgcacctgcc agccaggcct cggacgagga
     3961 ggacgcgccc gccaccgaca tctacttttg agtcgacggt accgcgggcc cgggatccac
     4021 cggatctaga taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt
     4081 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     4141 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4201 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4261 ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     4321 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     4381 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     4441 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     4501 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     4561 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     4621 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     4681 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     4741 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4801 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     4861 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     4921 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     4981 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     5041 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     5101 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     5161 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     5221 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     5281 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     5341 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     5401 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     5461 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     5521 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     5581 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     5641 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     5701 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     5761 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     5821 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     5881 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     5941 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     6001 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     6061 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     6121 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     6181 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     6241 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     6301 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     6361 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     6421 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     6481 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     6541 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6601 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
